Vibrio cholerae of serogroup 01 may induce severe diarrhoeal disease when multiplying in the gut of infected individuals by releasing cholera toxin (CT) which induces active electrolyte and water secretion from the intestinal epithelium. By analogous mechanisms several other bacteria, for instance Escherichia coli, may also cause diarrhoea by releasing other enterotoxins that may be related or unrelated to CT. CT is the prototype bacterial enterotoxin. It is a protein built from two types of subunits: a single A subunit of molecular weight 28,000 and five B subunits, each with a molecular weight of 11,600. The B subunits are aggregated in a ring by tight noncovalent bonds; the A subunit is linked to and probably partially inserted in the B pentamer ring through weaker noncovalent interactions. The two types of subunits have different roles in the intoxication process: the B subunits are responsible for cell binding and the A subunit for the direct toxic activity. The molecular aspects of toxin binding to intestinal and other mammalian cells and of the subsequent events leading to activation of adenylate cyclase through the intracellular action of the A subunit (and its A1 fragment) have been clarified in considerable detail (see J Holmgren, Nature 292:413-417, 1981). More recently information has also become available on the genetics and biochemistry of cholera toxin synthesis, assembly and secretion by V. cholerae bacteria. CT is encoded by chromosomal structural genes for the A and B subunits, respectively. These genes have been cloned from several strains, and their nucleotide sequences have been determined. The genes for the A and B subunits of CT are arranged in a single transcriptional unit with the A cistron (ctxA) preceeding the B cistron (ctxB). Studies on the organization of CT genes in V. cholerae strains of classical and El Tor biotypes have suggested that there are two copies of CT genes in classical biotype strains while there is only one copy in most El Tor strains (J. J. Mekalanos et al, Nature 306:551-557, 1983). The synthesis of CT is positively regulated by a gene, toxR that increases ctx expression manifold (V. L. Miller and J. J. Mekalanos, Proc Natl Acad Sci USA, 81:3471-3475, 1984). ToxR acts at the transcriptional level, and is present in strains of both classical and El Tor biotypes. ToxR probably increases ctx transcription by encoding a regulatory protein that interacts positively with the ctx promoter region. Studies on heat-labile enterotoxin (LT) in Escherichia coli (the subunit structure and function of LT is closely similar but not identical to CT) have shown that the A and B subunits are initially synthesized as precursors with a leader peptide preceeding the mature subunit proteins. These precursors are rapidly processed (i.e. the leader peptide is being removed) and translocated across the inner membrane into the periplasm, where unassembled monomeric B subunits pentamerize and associate with A subunit with a half-time of 1-2 min. The pathway of toxin assembly appears to proceed via A subunit association with B monomers or small oligomers. Once the complete toxin has assembled, in V. cholerae (in contrast to E. coli where the toxin remains in the periplasm the toxin is being translocated (secreted) across the V. cholerae 01 outer membrane through some sort of interaction of B subunit domains with the outer membrane (T. R. Hirst & J. Holmgren, Proc Natl Acad Sci USA, 84:7418-7422, 1987; S. J. S. Hardy et al, ibid, in press, 1988). If the B subunits of CT or LT are being expressed in the absence of any A subunit (several such strains have been prepared by chemical mutagenesis or deletions by recombinant DNA methods in the ctxA or eltA cistrons) the B subunits form pentamers which are then secreted from V. cholerae via the same pathway as for the intact toxin except for an apparently slightly slower assembly process in the periplasm (T. R. Hirst et al, Proc Natl Acad Sci USA 81:2645-2649, 1984; S. J. S. Hardy et al, ibid, in press, 1988). Because vaccination against cholera by parenteral injection has yielded only modest and short-term protection (usually less than 50% protection for less than 6 months), attention has turned to development of oral vaccines that stimulate intestinal immunity more efficiently. Special attention has been drawn to CTB pentamers as one component of such oral cholera vaccines (J. Holmgren et al., Nature 269:602-604, 1977). CTB is an effective oral immunizing agent which in a large field trial has been shown to afford protection against both cholera and diarrhoea caused by LT enterotoxigenic E. coli (J. Clemens et al, Lancet ii:124-127, 1986; J Infect Dis, in press, 1988). The separation of B subunit from A excludes any risk of reversion to toxicity, and CTB has been administered orally to more than 25,000 people without any side effects. These features have made CTB an important component, together with killed whole cholera vibrios, of a new oral cholera vaccine. Moreover, CTB has attracted much interest recently as an immunogenic carrier for various other peptide or carbohydrate antigens and has also been used as a receptor-blocking and receptor-modulating agent for short-term prophylaxis of cholera and E. coli diarrhoea (R. I. Glass et al, J Infect Dis 149:495-500, 1984; ST Donta et al, ibid 157:557-564, 1988; S. J. McKenzie and JF Halsey, J Immunol 133:1818-1824, 1984; A-M Svennerholm et al J Clin Microbiol 24:585-590, 1986).
These findings have emphasized a need to increase the yield of CTB for large-scale production, ideally avoiding at the same time the drawback in currently used preparation methods (see J. L. Tayot et al, Eur J Biochem 113:249-258, 1981) of having to purify the CTB protein from active toxin.
Therefore, with the aid of strategies and procedures described in this application we have constructed overexpression systems for CTB and CTB fusion proteins in which the CTB gene (or the gene for the hybrid fusion protein) is under control of strong foreign (non-cholera toxin) promoters. Our success in this regard contrasts with previous attempts by different procedures by J. J. Mekalanos et al (Nature 306:551-557, 1983) to attain this goal using one of the promoters (tacP) described in one of our examples, as these attempts were reported to fail since they resulted in expression of less CTB than achieved with the natural ctx promoter.